Correlation of addictive factors, human papilloma virusinfection and histopathology of oral submucous fibrosis

Ravi Mehrotra1, Ajay Kumar Chaudhary

2, Shruti Pandya

1, Sharmistha Debnath

1, Mangal Singh

3,

Mamta Singh1

1Department of Pathology, Moti Lal Nehru Medical College, Allahabad; 2Centre for Biotechnology, University of Allahabad,Allahabad; 3Otorhinolayngology, Moti Lal Nehru Medical College, Allahabad, India

BACKGROUND: Oral submucous fibrosis (OSMF) is a

widely prevalent, potentially malignant disorder of the

oral cavity in the Indian subcontinent. Areca nut has been

incriminated as the main causative factor. However,

other factors, including viruses may also play a role in the

initiation ⁄ development of these lesions. This study was

designed to assess the correlation of addictive factors,

human papilloma virus infection and histopathology of

OSMF.

METHODS: One hundred and thirteen cases of OSMF

were studied. Demographic information of each patient,

including the patient’s age, sex, addiction habits and his-

topathological grading was obtained. Presence of high-

risk human papilloma virus (HR-HPV) DNA was detected

using the Hybrid Capture II assay.

RESULTS: Of 105 cases finally included in the study, 33

(31.4%) patients were found to be positive for HR-HPV,

while 72 (68.6%) were negative. A total of 52 ⁄ 105 (49.5%)

patients chewed areca nut, of whom 18 (34.6%) patients

were found to be positive and 34 (63.5%) were negative

(P = 0.08). A total of 18 ⁄ 105 (17.1%) patients were ad-

dicted to smoking, of whom five (27.7%) were positive

(0.69). Only one (11.1%) patient, who was only habituated

to alcohol, was positive (1.08). Of the 105 patients 26

(24.7%) chewed as well as smoked tobacco, of whom, nine

(34.6%) were positive and 17(65.3%) were negative

(P = 0.67). Thus, there was no significant association be-

tween the various addiction habits and the presence of

HR-HPV. On correlating the various histopathological

grades of the disease with HR-HPV positivity a Pearson

correlation of 0.053 was obtained (P = 0.588). Thus, again

no significant correlation was observed between the dif-

ferent histopathological grades and HR-HPV positivity.

CONCLUSION: In conclusion, it was found that there is

neither significant correlation between HR-HPV infection

and the various addiction habits nor with the different

histopathological grades of the disease. To the best of our

knowledge, a study of these different factors has never

been attempted before in patients with OSMF.

J Oral Pathol Med (2010) 39: 460–464

Keywords: habits; histopathology; HR-HPV; oral submucous

fibrosis

Introduction

Oral submucous fibrosis (OSMF) is a potentially malig-nantdisorderoftheoralcavity–producingchangessimilarto those of scleroderma. It presents as a whitish-yellowdiscoloration involving the buccal mucosa, the orophar-ynx and rarely the larynx. It has a chronic, insidiousbiological course (1). Over the years, the incidenceof OSMF has increased manifold in India – especiallyamong the younger generation (2). Areca nut is the maincausative factor.Commercially freeze-driedproductssuchas paan masala, dohra, gutka and mava have a highconcentrationof areca nut andappear to causeOSMF(3).Other factors, including viruses, especially humanpapilloma virus (HPV), may also play a role in theinitiation or development of these lesions.

Human papilloma virus belongs to the family Papil-lomaviridae, has a small, non-enveloped DNA and caninduce hyperplasia, papillomatosis and verrucouslesions in the stratified squamous epithelium of the skinand mucosa (4). Over 100 different types of HPV havebeen reported in humans. Some of them, like HPVsubtypes 16, 18, 33 and 58 play an important role in thedevelopment of various kinds of tumors such as cervical,anogenital and head neck carcinoma (5). High-risk HPVhas also been detected in normal oral mucosa (6).At present, there is a paucity of information on thepotential role of HPV in potentially malignant orallesions. As OSMF is a common health problem in thispart of India (7), this study was designed to assess theprevalence of high-risk HPV and investigate its possiblerole in the etiology and development of this disease aswell as to correlate the presence of HPV with addictivehabits and histopathological grading of the disease.

Material and methods

One hundred and thirteen patients and 83 age andsex-matched controls from the Department of Otorhino-laryngology, Moti Lal Nehru Medical College,

Correspondence: Dr Ravi Mehrotra, Department of Pathology,M.L.N Medical College, Allahabad, India. Tel: +91 532 2256 472,Fax: +91 532 2256 507, E-mail: rm8509@gmail.comAccepted for publication September 1, 2009

J Oral Pathol Med (2010) 39: 460–464

ª 2010 The Authors. Journal compilation ª 2010 John Wiley & Sons A/S Æ All rights reserved

interscience.wiley.com/journal/jop

doi: 10.1111/j.1600-0714.2009.00849.x

Allahabad, India were studied from September 2007 toApril 2009, in a random manner after obtaining consentfrom the institutional ethical committee.

Detailed demographic information of each patient,including the patient’s age, sex, occupation, dietaryhabits and addiction habits was obtained. Emphasiswas placed on collecting information about addiction,education and ethnicity of subjects. Patients had noprevious history of treatment for HPV infection.

Detailed clinical examination of each patient wasperformed to assess the site, size and type of lesion. Forconfirmation of the clinical diagnosis, histopathologicalexamination was carried out. Patients with OSMF wereincluded and lesions with an abnormal epithelial surfacelike erythroplakia, leukoplakia and submucosal lesionsincluding hemangioma, mucocele, papilloma, aphthousulcers, melanoplakia and fibroma were excluded. Ascalpel biopsy tissue was performed as per standardprotocol and the tissue was processed by paraffinembedding 2–3 micrometer thick sections were cut andstained by hematoxylin and eosin (H and E). Biopsiesthat were inadequate on histopathology were excludedfrom the study. Histopathological examination wasperformed and results were recorded according to thetraditional grading of OSMF by Pindborg and Sirsat(8). Grading criteria used were as follows.

Very early stage (Grade I):

• A finely fibrillar collagen, dispersed with markededema.

• The fibroblastic response is strong.• The blood vessels are sometimes normal, but moreoften they are dilated and congested.

• Inflammatory cells, mainly polymorphonuclear leu-kocytes with an occasional eosinophil are present.

Early stage (Grade II):

• The juxta-epithelial area shows early hyalinization.• Plump young fibroblasts are present in moderatenumbers.

• The blood vessels are dilated and congested.• The inflammatory cells are mostly mononuclearlymphocytes, eosinophils and an occasional plasmacell.

Moderately advanced stage (Grade III):

• The collagen is moderately hyalinized.• The fibroblastic response is less marked, the cellspresent being mostly adult fibrocytes.

• Blood vessels are normal or constricted.• The inflammatory exudates consist of lymphocytesand plasma cells, although an occasional eosinophilis observed.

Advanced stage (Grade IV):

• The collagen is completely hyalinized.• The hyalinized areas are devoid of fibroblasts.• Blood vessels are completely obliterated or nar-rowed.

• The inflammatory cells are lymphocytes and plasmacells.

All specimens were examined independently by twodifferent histopathologists in a double blind fashion.If there were any discrepancies, a third opinion wasobtained and two similar opinions constituted the finaldiagnosis.

Samples were collected from the suspicious lesions ofthe oral cavity by a soft brush provided in the kit forHPV detection. Gentle rolling strokes were made overthe affected area as per the manufacturer’s instructionsand cytological samples were collected in the collectiontubes, which were then stored at 4�C until the Hybridcapture test was performed.

Detection of high-risk HPVSecond-generation commercial HPV test, the HybridCapture II assay Digene� (Digene Corporation,Gaithersburg, MD, USA), an FDA approved test forclinical diagnostics, was utilized. Detection of HPVDNA was carried out in the presence of a probe whichconsisted of the patented 13 HR-HPV genotypes (16, 18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). Thistechnique is a nucleic acid hybridization assay withsignal amplification that utilizes microplate chemilum-iniscent detection.

First, double stranded DNA was denatured by using astrong alkaline denaturation solution and converted intosingle stranded DNA (ssDNA). This ssDNA was thenhybridized in-solution to a cocktail of specific 13 HR-HPV RNA probes. The resultant DNA-RNA hybridsare captured onto the surface of a microwell platecoated with specific antibodies for DNA-RNA hybrids.The immobilized hybrids are then reacted with alkalinephosphatase-conjugated antibody and detected bycleavage of the chemiluminescent substrate. The lightsignals emitted were measured as relative light unit(RLU) in a luminometer (DML 2000, Digene�). Theintensity of the light was proportional to the amount oftarget DNA in the sample.

Estimation of HPV viral loadRatio of RLU of the sample to mean of RLU of threepositive controls (PC) was taken as an estimate ofapproximate viral load which relates to the index of theintensity of HPV infection. This ratio of any specimenrepresents empirically a relative measure of the viralload in it. Cut-off value (RLU of specimen ⁄mean RLUof PC) was 1.0 pg ⁄ml of sample.

Interpretation of cut-off value (RLU of specimen ⁄meanRLU of PC)Cut-off ratios of 0 to 0.99 were negative for HR HPV;Cut-off ratio >1.00 was considered positive for HRHPV (Table S1). The cut-off ratio of 1.0 correspondedto viral DNA load of 5000 copies ⁄ml of 1 pg ⁄ml at athreshold of finding a clinical disease or prognosis of apotentially malignant lesion.

Statistical analysisThe odds ratio (OR) and 95% confidence interval (CI)were calculated using SPSS12.0 software package (SPSSCompany, Chicago, IL, USA).

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Results

Of the original 113 patients, the brush samples of 105patients were found to be adequate. Demographicdetails are shown in Table 1.On analyzing the HR-HPV results, in the patients’

samples, 33 (31.4%) were positive and 72 (68.6%) werenegative, while in 83 control samples, 16 (19.3%) werepositive and 67 (80.7%) were negative. A total of 52 ⁄ 105(49.5%) patients chewed areca nut, of whom 18 (34.6%)patients were found to be HR-HPV positive and 34

(63.5%) were negative. (OR = 2.74, 95% CI = 0.94–4.86 P = 0.08). Of 105, 18 (17.1%) patients wereaddicted to smoking, of whom, five (27.7%) werepositive. (OR = 1.92, 95% CI = 0.38–9.64 P = 0.69)Only one (11.1%) patient, who was habituated only toalcohol, was positive. (OR = 0.38, 95% CI = 0.01–8.10 P = 1.08). A total of 26 ⁄ 105 (24.7%) patientschewed as well as smoked tobacco, of these nine (34.6%)were positive and 17 (65.3%) were negative. (OR =1.58, 95% CI = 0.46–5.24 P = 0.67). (Table 2) Thus,there was no significant association between the variousaddiction habits and the presence of HR-HPV.

On histopathological grading of the biopsies, 24(22.8%), 38 (36.2%) and 43 (40.9%) patients hadOSMF Grade I, II and III respectively. In OSMF gradeI, 12 patients were areca nut chewers, six were tobaccosmokers and two drank alcohol, while four wereaddicted to both tobacco smoking and areca nutchewing. Of these seven (29.1%) patients were HR-HPV positive and 17 were negative. In OSMF grade II,17 patients were addicted to areca nut ⁄ commerciallyavailable products containing areca nut, seven weresmokers, and four took alcohol, while 10 patients wereaddicted to both areca nut and tobacco. A total of 11 ⁄ 38showed HR-HPV positivity, while 27 were negative. Inthe histopathological grade III category, 23 patientswere addicted to areca nut, five were smokers and threewere habituated to alcohol, while 12 consumed bothtobacco and areca nut. A total of 15 ⁄ 43 were positive

Table 1 Demographic distribution of patients and controls

Patients history Totalnumber ofcontrols83 (%)

Totalnumberof cases105 (%)Variable Categories

Gender Male 57 68.6 74 70.4Female 26 31.3 31 29.5

Age(years)

<20 19 22.8 16 15.221–40 31 37.3 54 51.441–60 27 32.5 23 21.9>61 6 7.2 12 11.4

Education Illiterate 39 46.9 51 48.5Literate 44 53 54 51.4

EthnicGroup

Hindu 63 75.9 61 58Muslim 17 20.4 33 31.4Others 3 3.6 11 10.4

Table 2 Distribution of patients and control subjects according to HPV infection with addiction

Addiction habit

Totalnumber

of controls83

HPV +vecontrols16 (19.3)

HPV )vecontrols67 (80.7)

Totalnumberof cases105

HPV +vecases

33 (31.4)

HPV )vecases

72 (68.6) OR 95% CI P value

Areca nut chewing 7 (44) 6 (16.2) 31 (83.7) 52 (49.5) 18 (34.6) 34 (63.5) 2.74 0.9381–4.8572 0.08Tobacco smoking 18 (21.6) 3 (16.6) 15 (83.3) 18 (17.1) 5 (27.7) 13 (72.2) 1.92 0.38–9.64 0.69Alcohol 4 (4.8) 1 (25) 3 (75) 9 (8.5) 1 (11.1) 8 (88.8) 0.38 0.01–8.10 1.08Tobacco smoking andareca nut chewing

24 (28.9) 6 (25) 18 (75) 26 (24.7) 9 (34.6) 17 (65.3) 1.58 0.46–5.42 0.67

Values are given as n (%).

12

17

23

6 75

24 34

1012

7

11

1517

2728

0

5

10

15

20

25

30

OSMF I (24) OSMF II (38) OSMF III (43)

Areca nutchewing

Smoking

Alcohol

Tobacco smokingand areca nutchewingHPV positive

HPV negative

Graph 1 Distribution of the various risk factors in OSMF Grade I, II and III.

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for HR- HPV and 28 were negative (Graph 1). Oncorrelating OSMF grading I. II and III with HR-HPVpositivity, Pearson correlation of 0.053 was obtained(P = 0.588). Thus, no significant correlation wasobserved between the different grades of the diseaseand HR-HPV positivity.

Discussion

Oral submucous fibrosis is a potentially malignantdisorder of oral cavity and is commonly seen in clinicalpractice, at times, coexisting with oral cancer (9). Thisdisease occurs most commonly in South Asia. In India,about 5 million people suffer from OSMF (10). Of the105 patients finally included in this study, 74 (70.4%)were males and 31 (29.5%) were females, yielding a maleto female ratio of 2.3:1. Similarly, Ahmed et al., (11) intheir study from Patna, Bihar, also reported a male:female ratio of 2.7:1. However, Hazarey et al. (12) fromNagpur, Central India reported a male to female ratio of4.9:1. It is likely that the areca nut chewing habits inAllahabad and Patna, both situated in North India aremore similar vis-a-vis in Nagpur in Central India –where a marked male predominance was noted.

In this study, maximum number of cases belonged tothe 21–40 year age group with a mean age of 30 yearsand most of them were in the low or middle socioeco-nomic group. Kumar et al. found similar results fromChennai. The chewing habit of areca nut without anytobacco and the use of various commercial productscontaining areca nut were significant risk factors forOSMF (13).

In this study, areca nut chewing was found to be themost frequent addiction i.e. 52 (49.5%), followed by 26(24.7%) patients who both smoked tobacco and chewedareca nut. On this basis, it was found that only arecachewers had more OSMF cases compared with thosewho both smoked and chewed. In a recent study, wereported lack of correlation between habits and clinicalfindings in patients with OSMF (14). A recent studyfrom Taiwan reported that 36.4% patients of OSMFwere addicted to betel quid chewing and 45.5% weresmokers (1). In this country, patients with OSMFusually consume gutka (mixture of areca nut, tobaccoand condiments) (2). Excessive use of paan masala andgutka containing areca nut may affect the buccalmucosa of the oral cavity and the combination of arecanut and tobacco may have an additive role to play in theinitiation of the disease.

In this study, 33 (31.4%) patients were reportedpositive for HR-HPV while 72 (68.6%) cases were foundnegative. On the other hand, in the control group,16 (19.3%) subjects were found positive while67 (80.7%) were negative. Kujan et al., (15) however,reported that all oral samples from control individualsfrom United Kingdom were negative for HR-HPV usingthe same technique. This could be resulting from ahigher background positivity of HR-HPV in India vis-a-vis countries like United Kingdom. Vidal et al. fromBrazil recently studied the role of HPV by this assay inpatients with oral carcinoma and reported 22.5%

positivity of high risk-HPV. They also suggested thatthe detection of HPV by this technique not only helpedto identify viral infections, but also corresponded tofindings of koilocytosis on exfoliative cytology andconcluded that presence of HPV might also contributeto the development of cancer, but there may be manyother factors such as smoking, tobacco chewing andradiation that were consistently present in personssuffering from oral malignancies (16).

A significant association was not found between thevarious addiction habits and the presence of HR-HPV.The correlation of HPV positivity with various addic-tion habits, to the best of our knowledge, has never beenattempted before in patients with OSMF.

On correlating histopathological grading with HPVpositivity, 29.1% patients of grade I, 28.9% of grade IIand 34.5% of grade III were positive for HR-HPV(Graph 1), Pearson correlation of 0.053 was obtained(P = 0.588) and no significant correlation was observedbetween the different grades of the disease and HR-HPVpositivity. We have recently reported the correlation ofhistopathological grading and addiction habits andfound that maximum patients (46.8%) were in gradeIII who took tobacco products for 8–10 years or morewith high frequency (7–10 times ⁄ day) (14).

Although areca nut use has been the most frequentlyreported causative factor for OSMF, the role of HPV, ifany, in the disease initiation or progression is still notclear. Understanding the role of HPV in oral carcinogen-esis is complicated by widely varying reported frequencyof HPV infection (0–100%) in potentially malignantlesions and oral malignancies, probably as a result ofdifferences in sampling methods (biopsy, washings, andbrushings), patient profile, sensitivity of diagnostic meth-ods (in situ hybridization, polymerase chain reaction etc)(17, 18). D’Costa et al. performed typing of HPV fromBombay by Southern hybridization analysis of PCRproducts using HPV16, 18, 33 6 and 11 type specificoligonucleotide probes and reported 31% of normalmucosa, 34% potentially malignant lesions and 15% oralcarcinoma were positive for HPV 16, 18 and 33; however,low-risk HPV 6–11 were not detected in any of the orallesions (19). In this study, however, we did not attempt toclassify theHPV subtypes. Further studies are planned byusing different molecular techniques to further determinethe subtypes of oncogenic HPV.

Conclusions

In conclusion, it was found that there is no significantcorrelation between HR-HPV infection and the variousaddiction habits or with the different histopathologicalgrades of the disease. To the best of our knowledge, astudy of these different factors has never been attemptedbefore in patients with OSMF. Keeping in view, therecent advent of the HPV vaccine and its availability forclinical use, the significance of HPV in different orallesions cannot be undermined. Possible universal immu-nization of all adolescents in the future by the vaccine,although an utopic thought at present, may sound thedeath knell of HPV related potentially malignant and

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malignant lesions both oral and cervical. Investigationon the role of HPV markers could be rewarding inplanning long-term strategies for prevention, diagnosisand possible cure of these conditions. These findingswarrant further study with a larger number of patients.

References

1. Chen PC, Pan CC, Kuo C. Risk of oral nonmalignantlesions associated with human papillomavirus infection,betel quid chewing, and cigarette smoking in Taiwan: anintegrated molecular and epidemiologic study. Arch PatholLab Med 2006; 130: 57–61.

2. Gupta PC, Sinor PN, Bhonsle RB. Oral submucousfibrosis in India: a new epidemic. Natl Med J India 1998;11: 113–6.

3. Tilakaratne WM, Klinikowski MF, Saku T, et al. Oralsubmucous fibrosis: review on aetiology and pathogenesis.Oral Oncol 2005; 30: 30–7.

4. Scully C. Oral squamous cell carcinoma: from a hypoth-esis about a virus, to concern about possible sexualtransmission. Oral Oncol 2002; 38: 227–34.

5. Chaudhary AK, Singh M, Sundaram S, Mehrotra R. Roleof human papillomavirus and its detection in potentiallymalignant and malignant head and neck lesions: updatedreview. Head Neck Oncol 2009; 1: 22.

6. Terai M, Hashimoto K, Yoda KP. High prevalence ofhuman papillomaviruses in the normal oral cavity ofadults. Oral Microbiol Immunol 1999; 14: 201–5.

7. Mehrotra R, Pandya S, Chaudhary AK, et al. Prevalenceof oral pre-malignant and malignant lesions at a tertiarylevel hospital in Allahabad, India. Asian Pac J CancerPrev 2008; 9: 263–6.

8. Pindborg JJ, Sirsat SM. Oral submucous fibrosis. OralSurg Oral Med Oral Pathol 1966; 22: 764–79.

9. Ho PS, Yang YH, Shieh TY. Consumption of areca quid,cigarettes, and alcohol related to the comorbidity of oralsubmucous fibrosis and oral cancer. Oral Surg Oral MedOral Pathol Oral Radiol Endod 2007; 104: 647–52.

10. Chiu CJ, Chang ML, Chiang CP. Interaction of collagen-related genes and susceptibility to betel quid induced oralsubmucous fibrosis. Cancer Epidemiol Biomarkers Prev2002; 11: 646–53.

11. Ahmed MS, Ali SA, Ali AS. Epidemiological andetiological study of oral submucous fibrosis among gutkhachewers of Patna, Bihar, India. J Oral Soc PedodonticsPrev Dent 2006; 24: 84–9.

12. Hazarey VK, Erlewad DM, Mundhe KA. Oral submucousfibrosis: study of 1000 cases from Central India. J OralPathol Med 2006; 35: 1–6.

13. Kumar KK, Saraswathi TR, Ranganathan K, et al. Oralsubmucous fibrosis. A clinico-histopathological study inChennai. Indian J Dent Res 2007; 18: 106–11.

14. Pandya S, Chaudhary AK, Singh M, et al. Correlation ofhistopathological diagnosis with habits and clinical find-ings in oral submucous fibrosis. Head Neck Oncol 2009; 1:10.

15. Kujan O, Desai M, Sargent A. Potential application oforal brush cytology with liquid-based technology: resultfrom a cohort of normal oral mucosa. Oral Oncol 2006; 42:810–8.

16. Vidal A, Junior A, Mello R. HPV detection in oralcarcinomas. J Bras Pathol Med Lab 2004; 40: 21–6.

17. Miller CS, Johnstone BM. Human papillomavirus as arisk factor for oral squamous cell carcinoma: a meta-analysis, 1982–1997. Oral Surg Oral Med Oral Pathol OralRadiol Endod 2001; 91: 622–35.

18. Uobe K, Masuno K, Fang Y. Detection of HPV inJapanese and Chinese oral carcinomas by in situ PCR.Oral Oncol 2001; 37: 146–52.

19. D’costa J, Saranath D, Dedhia P. Detection of HPV-16genome in human oral cancers and potentially malignantlesions from India. Oral Oncol 1998; 34: 413–20.

Acknowledgements

The authors thank the Department of Science and Technology (DST), New

Delhi for providing financial support (Grant No.SR ⁄ SO ⁄HS-127 ⁄ 2007) toAKC for this study. They also thank Dr. Atul Kr Singh, Associate

Professor, Allahabad Degree College, University of Allahabad for help in

statistical analysis of the results.

Competing interests

The authors declare that they have no competing interests.

Supporting information

Additional Supporting Information may be found in theonline version of this article:

Table S1 Quantitative data of each patient depictingHPV RLU and Histopathological grading. (For onlineversion)

Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.

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